In biological studies, it is important to distinguish between live and dead cells. This is important for situations in which cells: are in cellular suspensions, are grown on a substrate, are in tissue that is removed from the body or are in tissue in the body. For instance it is important to know prior to the study of a certain manipulation of the cells outside or inside the body if they are alive or not. This manipulation can be of different types such as: mechanical or electrical or chemical. For instance, when a drug or a chemical is tested on cells in suspension, or on substrates or in tissue it is important to know if the cells were alive prior to the test. Otherwise it is impossible to determine the effect of the drug or the chemical. Many times after the manipulation it is important to know if the cells are alive or not. For instance after chemical drug treatment of cancer it is important to know if the drug has killed the cells or not. Or for instance after a mechanical injury it is important to know if the traumatized cells are alive or not. Or for instance after thermal treatment of cancer with such methods as focused ultrasound, hyperthermia, electroporation or cryosurgery it is important to know if the treated cells are alive or not. In regular accidents it is important to know if the cells are alive for instance after the body was exposed to hypothermia or hypoxia. Or, after a stroke it is important to know what is the extent of tissue damage in the brain.
There are essentially two ways in which the viability of the cell can be determined. One is through their function and the other through their properties. An example of how to test viability through their function is by measuring their metabolism. For instance their ability to process glucose is used with positron emission tomography (PET) measurements. One of the most common methods to determine cell viability through their properties is by determination of the cell membrane integrity. The cell membrane has evolved to separate between the intracellular milieu and the extracellular. The function of the cell membrane is to maintain an intracellular environment compatible with the optimal function of the intracellular components and not to allow undesirable chemical to enter the cell interior. One of the main features separating dead from live cells is the loss of the physical integrity of the plasma membrane (Darzynkiewicz, Z., Li, X., and Gong, J. P. (1994) in Methods in Cell Biology Academic Press, Inc., New York; King, M. A. (2000) J. Immunol. Methods 243, 3-12.) When the membrane integrity is lost, chemicals that would otherwise not enter the cell can enter. Therefore, a variety of viability tests have been designed which test if chemical that cannot penetrate the membrane of intact cells, are inside the cells. The most common such chemicals are colorimetric dyes such as trypan blue and fluorescent dyes such as propidium iodide or YOYO-1 (Molecular Probes OR), which change the cell color once inside the cell (Horan, P. K., and Kapler, J. W. (1977) J. Immunol Methods 18, 309-316; Shapiro, H. M. (1995) in Practical Flow Cytometry Wiley, New York; Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.) These chemicals are commonly used to determine cell viability in cells in suspension (Rui, J., Tatsutani, K. N., Dahiya, R., Rubinsky, B. Effect of thermal variables on human breast cancer in cryosurgery. Breast Cancer Research and Treatment, 53 182-192, 1999) as well as cells in tissue that was excised from the body (Pham, L., Rubinsky, B., “Breast tissue cryosurgery with antifreeze proteins” HTD-Vol. 362/BED-Vol. 40, Advances in Heat and Mass Transfer in Biotechnology—ASME Press, pp 171-175. 1998). However, the use of these colorimetric or fluorescent methods to detect cell viability through the measurement of the cell membrane integrity have certain drawbacks. First of all the methods require a first step that is the adding of a discrimination agents to the cell suspensions or to the tissue. Second, a certain period of time is needed for the cell to incorporate the discriminating chemical. Third, the presence of the discriminating chemical inside the cell must be determined through the use of cumbersome optical inspection apparatus, such as microscope or flow cytometry devices. These devices require direct interaction with the tested cells. Fourth, though widely used in biology and clinical research, the dye-exclusion based assays lack the capability to produce instantaneous and quantitative cell viability information, which is particularly important in the study of dynamics of cell death.
It is the purpose of the present invention to provide a cell viability assay that tests for the cell membrane integrity in a way that resolves drawbacks with the colorimetric or fluorescent dyes, listed above.